The goal is to develop a suite of rapid, reliable, semiautomated DNA probe assays for antibiotic-resistant Staphylococcus species responsible for a variety of nosocomial infections. First, we will develop a DNA probe test for the mecA gene of methicillin-resistant S. aureus (MRSA) and coagulase- negative Staphylococcus species (MRCNS). The mecA gene codes for methicillin resistance in 97% to 100% of MRSA and MRCNS isolates. Direct detection of mecA will be more rapid, accurate, and reliable than current culture-dependent methicillin susceptibility tests, which are compromised by highly variable expression of the resistance phenotype in vitro. The DNA probe test will also include probes for identification of individual Staphylococcus species, allowing simultaneous species ID and detection of methicillin resistance in a single test. The test will be conducted on colonies picked from primary isolation plates, and will require only 15 minutes of manual processing followed by 35 minutes of automated processing, yielding results in under 1 hour. We will also develop assays for resistance to second-line antibiotics used to treat MRSA and MRCNS infections. These tests will be conducted on isolates confirmed to be methicillin resistant by the combined mecA/species assay, and will require 2-4 hours to obtain results with under 30 minutes of manual processing. The entire suite of assays will yield complete species ID and antibiotic susceptibility results within half a day of picking colonies from plates.